Transformation and foreign gene expression in brassica species

ABSTRACT

Brassica species are produced by transformation of cell cultures with foreign DNA followed by regeneration of plants from transformed cells. The cells and the plants produced thereby are capable of expressing the foreign gene. The Brassica species are transformed employing a manipulated Agrobacterium transformation system, followed by regeneration of the plant tissue into plants.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 054,187, filed May 26,1987, now abandoned, which is a continuation-in-part of application Ser.No. 868,640, filed May 29, 1986, now abandoned.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention is related to a method for improving genotypes andassociated phenotypes in Brassica species by means ofAgrobacterium-based genetic transformation.

BACKGROUND

Cruciferous species of the tribe Brassiceae are widely used as a sourceof protein, oil, condiments and chemical feedstocks. Significant efforthas been expended to improve the quality of cultivated Brassica speciesby conventional plant breeding, and a number of major successes arerecorded. The methods of conventional plant breeding have been limited,however, to the movement of genes and traits from members of the genusBrassica to others of the same genus and, in a few notable examples,"wide crosses" from other closely related genera. The development of amethod for introducing foreign genes into Brassica species would greatlyenhance the range of traits which could be imparted to Brassica oilseedsand vegetables.

In order to obtain a reliable system for useful gene introduction intoBrassica species, a number of obstacles must be overcome. These includeoptimization of regenerability to whole plants of the target tissue,definition of the conditions (e.g., time, bacterial concentration, andmedia) for the co-cultivation of the Brassica cells and Agrobacteriumcells, discovery of an Agrobacterium strain of suitable virulence withBrassica for gene transfer, identification of a suitable regulatorysequence (promoter) to ensure expression of the foreign gene in thetransformed tissue, and expression of a selectable marker enablingidentification of transformants.

Brief Description of Relevant Literature

Brassica species have been widely investigated for regenerability fromtissue explants. Both Brassica napus and Brassica oleracea show shootregeneration from a variety of tissues including hypocotyls (M. F.Dietert et al., Plant Science Letters (1982) 26:233-240), leaf callus(G. R. Stringham, Z. Pflanzenphysiol. (1979) 92:459-462), roots (P. A.Lazzeri and J. M. Dunwell, Annals of Botany (1984) 54:341-350), and leafand stem protoplasts (L. C. Li and H. W. Kohlenbach, Plant Cell Reports(1982) 1:209-211; K. Glimelius, Physiol. Plant. (1984) 61:38-44). Seealso, the poster given by Radke, et al., Crucifer Genetics Workshop,Guelph, June 29, 1986.

The suitability of Agrobacterium as a vector for Brassica transformationis suggested by host range studies by DeCleene and DeLey (Botanical Rev.(1976) 42:386-466) demonstrating several species of Brassica (includingnapus, oleracea, nigra and campestris) to be susceptible toAgrobacterium. Recent studies by L. A. Holbrook and B. L. Miki (PlantCell Reports (1985) 4:329-332) show some evidence for the expression ofcharacteristic Agrobacterium genes in non-regenerable tumorous tissue.

The use of Agrobacterium tumefaciens for transforming plants usingtissue explants is described in Horsch et al. (Science (1985)228:1229-1231). See also, Herrera-Estrella et al. (Nature (1983)303:209-213), Fraley et al. (Proc. Natl. Acad. Sci. USA (1983)80:4803-4807) and Bevan et al. (Nature (1983) 304:184-187). Use of the35S promoter from cauliflower mosaic virus to direct expression ofchimeric genes in plants has been reported (see C. K. Shewmaker et al.,Virology (1985) 140:281-288 and R. C. Gardner et al., Plant MolecularBiology (1986) 6:221-228).

SUMMARY OF THE INVENTION

Transformed Brassica plants and tissues are provided which contain novelnucleotide constructions capable of stable expression. Thetransformation techniques employed are designed to optimize frequency oftransformation, recovery of target tissue and regeneration of plantsfrom the transformed tissue. Preferred techniques of the invention forobtaining the desired transformed Brassica plants include use ofAgrobacterium tumefaciens strains (having virulence against Brassica),use of a medium during the selection process containing a less thannormal carbon-source content (less than or equal to 2% sucrose or itsequivalent in caloric value), use of efficient promoters, and use ofhypocotyls as target tissues. The technique may make use of a feederlayer of tobacco suspension cells to assist with transformationfrequency and recovery of the target tissue. Target tissue may includeleaf or stem explants in addition to the hypocotyls discussed above.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an autoradiography map created by polyacrylamide gelelectrophoresis of cellular protein to demonstrate the presence of theneomycin phosphotransferase gene in transformed cells growing onselective media by showing the existence of ATP-mediated phosphorylationof kanamycin using γ-³² P-labelled ATP: Lanes 1, 2, 3 and 4: Brassicanapus cv Westar tissue transformed by A281×200 Agrobacteriumtumefaciens. Lanes 5 and 6: Brassica napus cv Westar tissue transformedby K12×200 Agrobacterium tumefaciens. Lane 7: Negative controluntransformed Brassica leaf tissue. Lane 8: Bacterial neomycinphosphotransferase activity (positive control).

FIG. 2 is a graph showing sensitivity of transformed and untransformedBrassica tissue to kanamycin.

FIGS. 3A-3E are a pTiA6 T-DNA map and pathway to pTiK61.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Novel procedures and products are provided involving the introduction ofnovel nucleotide constructs into cells of Brassica napus, typicallyusing leaf and hypocotyl explants, where the transformed cells of theplant express one or more genes present in the construct so as toprovide at least one novel property for the plant, particularly aphenotypic property.

A number of process steps are involved in providing for efficientfrequency of transformation, recovery of target tissue, and regenerationof plants from the transformed tissue. The initial stage is theselection of an Agrobacterium tumefaciens strain which provides forefficient transformation of Brassica. In the next stage, selection andregeneration media are employed with only a small amount ofcarbon-source materials (less than 2% sucrose or its equivalent incaloric value). The construct which is used should include atranscriptional initiation region which efficiently functions inBrassica. The source of the plant cells for transformation is desirablyfrom hypocotyls.

Agrobacterium Strains

Although any of the numerous strains of Agrobacterium tumefacienscapable of transferring genetic material to Brassica species can be usedin combination with the other variations of the present invention,particularly improved transformation, recovery, and regeneration can beachieved by using Agrobacterium tumefaciens strains A281, EHA101, andK61, as well as other strains sharing common characteristics with thesestrains. These bacterial strains containing preferred plasmids(described later in detail) have been deposited with the American TypeCulture Collection, Rockville, Md., and granted ATCC Deposit AccessionNo. A. tumefaciens A281 (pCGN200) ATCC No. 67121; A. tumefaciens (K61),ATCC No. 53621.

A number of Agrobacterium tumefaciens strains have been developed havingdifferent Ti-plasmids, e.g., agropine or nopaline specific. Both armedand disarmed plasmids are employed, that is, the armed plasmids containoncogenic T-DNA that can be transferred to the plant cell and thedisarmed plasmids contain no oncogenes that can be transferred to theplant cell. The strains include Agrobacterium tumefaciens strains A281,EHA101, and K61.

Agrobacterium A281, containing the Ti-plasmid from strain Bo542, has thefollowing characteristics: biotype 1, agropine synthase and agropinedatabolism positive, and 3-ketolactose positive. This armed strain wasconstructed by an in planta conjugation of wild-type Bo542 with strainA136, a C58 nopaline strain derivative cured of its Ti-plasmid and maderesistant to rifampicin and nalidixic acid (E. Hood et al.,Biotechnology (1984) 2:702-708; D. Sciaky et al., Plasmid (1978)1:238-253). The virulence plasmid pTiBo542 is 249 kD that contains anintact vir region and T-DNA region.

Strain EHA101 is described in Hood et al., J. of Bacteriology (1986)168:1291-1301.

Strain K61 is derived from Agrobacterium strain K12 by tripartite matingwith pCGN567 containing the wide host range replication system pVK102(Knauf and Nester, Plasmid (1982) 8:45-54) and the left and right T-DNAborders of pTi86.

Agrobacterium strain A348, (Garfinkel et al., Cell (1981) 27:143-153)containing pTiA6 was transformed into strain A114 or NT1 and theresulting strain selected for octopine catabolism on BTB media (Hooykaaset al., J. Gen. Microbiol. (1979) 110:99-109) was named K12.

The Agrobacterium to be employed as the transformation system isconveniently transformed with a wide-host-range plasmid that can shuttleDNA from E. coli into Agrobacterium. This is achieved by having a P-1incompatibility plasmid replicon, e.g., RK2, and a plasmid repliconcapable of providing multicopies in E. coli, usually at least 5,preferably at least 10, and up to 200 copies in E. coli. Thewide-host-range plasmid will be characterized by having at least oneT-DNA border sequence, particularly the right border sequence, orconveniently having both border sequences separated in one direction bythe various constructs intended to be integrated into the plant speciesgenome. The Agrobacterium strain may have either a disarmed Ti- orRi-plasmid, as indicated above. The plasmid pCGN200 can be transformedinto A. tumefaciens and detected by kanamycin resistance. Plant cellsmay then be cocultivated with the A. tumefaciens transformant, grown andselected for resistance to a biocide and expression of the desiredgene(s) and can be monitored by Southern and Western blots,immunoassays, and the like. Of particular interest as markers aremarkers which impart biocide resistance to plant cells and plants, sothat the transformed plant species can be efficiently selected.

The Transformation Process

The transformed plant cells may be cells in culture, cells present as adisorganized mass in a callus, cells organized as leaf explants, shootcultures, seeds, fruits, leaves, roots, or cells organized as a wholeplant. Hypocotyl segments are particularly preferred as target cells forforming the transformed plant cells as an enhanced transformation andrecovery rate results from the use of hypocotyl segments. A hypocotyl isthe part of the axis, or stem, below the cotyledons in the embryo of aplant.

The Agrobacterium strain will include on a plasmid, either theTi-plasmid or the wide host range plasmid, a foreign construct, whichforeign construct is destined to be transferred to the plant cell. As aresult of such transfer, the foreign construct, will normally be presentin all or substantially all of the cells of the plant tissue aftertransformation and regeneration, but expression may be limited toparticular cells or particular stages in the development of the plant.The foreign construct will include transcriptional and translationalinitiation and termination signals, with the initiation signals 5' tothe gene of interest and the termination signals 3' to the gene ofinterest in the direction of transcription.

The transcriptional initiation region which includes the RNA polymerasebinding site (promoter) may be native to the plant host or may bederived from an alternative source, where the region is functional inthe Brassica host. A wide variety of transcriptional initiation regionsmay be employed, including those which are endogenous to Brassica orspecific Brassica species, e.g. nupus, or exogenous to Brassica, thatis, comes from a cellular source other than a Brassica species cell. Thesources of such transcriptional initiation regions may include otherplant species, plant viruses, and bacterial plasmids, such as the Ti- orRi-plasmids, particularly the T-DNA genes which are functional in plantcells. The transcriptional initiation regions may be constitutive orregulatable. Regulatable genes may be regulatable by external signals,including physical signals such as light and heat, chemical signals,such as metabolites, or cell differentiation signals, such asroot-specific, leaf-specific, seed-specific, etc., or stress-relatedsignals, etc. A preferred promoter region is the 35S promoter fromcauliflower mosaic virus. This promoter is well known but has not beenused previously with Brassica.

The 3' termination region may be derived from the same gene as thetranscriptional initiation region or a different gene. For example,where the gene of interest has a transcriptional termination regionfunctional in a Brassica species, that region may be retained with thegene.

An expression cassette can be constructed which will include thetranscriptional initiation region, the gene of interest under thetranscriptional regulational control of the transcriptional initiationregion, the initiation codon, the coding sequence of the gene (with orwithout introns), and the translational stop codons, and will befollowed by the transcriptional termination region (which will includethe terminator and may include a polyadenylation signal sequence andother sequences associated with transcriptional termination). Thedirection is 5'-3' in the direction of transcription. The cassette willusually be less than about 10 kD, frequently less than about 6 kD,usually being at least about 1 kD, more usually being at least about 2kD.

The gene of interest may be derived from a chromosomal gene, cDNA, asynthetic gene, or combinations thereof. Where the expression product ofthe gene is to be located in other than the cytoplasm, the gene willusually be constructed to include particular amino acid sequences whichresult in translocation of the product to a particular site, which maybe an organelle (such as the chloroplast, mitochondrion or nucleus) orthe cell plasma membrane, or the product may be secreted into theperiplasmic space or into the external environment of the cell. Varioussecretory leaders, membrane integrator sequences, and translocationsequences (transit peptides) for directing the peptide expressionproduct to a particular site are described in the literature. See, forexample, Cashmore et al., Biotechnology (1985) 3:803-808; Wickner andLodish, Science (1985) 230:400-407.

Genes of interest for use in Brassica species include a wide variety ofphenotypic and non-phenotypic properties. Among the phenotypicproperties are enzymes which provide for resistance to stress, such asdehydration resulting from heat and salinity, resistance to insects,herbicides, toxic metal or trace elements, or the like. Resistance maybe as a result of a change in the target site, an enhancement of theamount of the target protein in the host cell, an increase in one ormore enzymes involved with the biosynthetic pathway to a product whichprotects the host against the stress, or the like. Genes may be obtainedfrom prokaryotes or eukaryotes, including but not limited to bacteria,fungi (e.g., yeast), viruses, plants, or mammals or may be synthesizedin whole or in part. Illustrative genes include glyphosate resistant3-enolpyruvylphosphoshikimate synthase gene, nitrilase, genes in theproline and glutamine biosynthetic pathway, and metallothioneins. Othergenes of interest may be involved with regulation of growth, such asmanipulations of source/sink (carbon partitioning) relations, hormonalregulation; resistance to herbicides, such as phenmedipham; productionof male sterility; regulation of photosynthetic efficiency, such asaltering the efficiency of RuBP carboxylase; control of the quality ofthe plant taste or nutritional value; altering oil or protein profile,yield, or quality or reduction of specific undesirable metabolites suchas glucosinolates or extremely long chain fatty acids e.g., C₂₂ fattyacids.

Instead of an expression cassette, one may have a transcriptioncassette, where the RNA sequence which is produced is complementary toan endogenous transcriptional product. The complementary or antisensesequence may be to an open reading frame, or a non-coding region, suchas an intron or a 5'-non-coding leader sequence. In this way, theexpression of various endogenous products may be modulated.

One or more cassettes may be involved, where the cassettes may beemployed in tandem for the expression of independent genes which mayexpress products independently of each other or may be regulatedconcurrently, where the products may act independently or inconjunction.

Where the expression cassette is to be transformed into plant cells bymeans of Agrobacterium, the cassette will be bordered by the right andoptionally left T-DNA borders. These borders may be obtained from anyTi- or Ri-plasmid and may be joined to the expression cassette byconventional means. The expression cassette may be constructed so as tobe directly transferred from a plasmid other than a Ti- or Ri-plasmid ormay become integrated into the T-DNA region of a Ti- or Ri-plasmidthrough homologous recombination. Thus, the expression cassette couldhave DNA sequences at one or both borders of the expression cassettehomologous with sequences present in the T-DNA region of the Ti- orRi-plasmid.

The expression cassette will normally be carried on a vector having atleast one replication system. For convenience, it is common to have areplication system functional in E. coli, such as ColE1, pSC101,pACYC184, or the like. In this manner, at each stage after eachmanipulation the resulting construct may be cloned and sequenced, andthe correctness of the manipulation can be determined. In addition to orin place of the E. coli replication system, a broad host rangereplication system may be employed, such as the replication systems ofthe P-1 incompatibility plasmids; e.g., pRK290. These plasmids areparticularly effective with armed and disarmed Ti-plasmids for transferof T-DNA to the plant species host.

In addition to the replication system, there will frequently be at leastone marker present, which may be useful in one or more hosts, ordifferent markers for individual hosts. That is, one marker may beemployed for selection in a prokaryotic host, while another marker maybe employed for selection in a eukaryotic host, particularly the plantspecies host. The markers may be protection against a biocide, such asantibiotics, toxins, heavy metals, or the like, or may function bycomplementation, imparting prototrophy to an auxotrophic host. Variousgenes which may be employed include neomycin phosphotransferase (NPTII;also known as APHII), hygromycin phosphotransferase (HPT),chloramphenicol acetyltransferase (CAT), nitrilase, and gentamicinresistance genes. For plant host selection, markers of particularinterest include NPTII, providing kanamycin resistance or G418resistance; HPT, providing hygromycin resistance; CAT, providingchloramphenicol resistance; mutated AroA gene, providing glyphosateresistance; etc.

The various fragments comprising the various constructs, expressioncassettes, markers, and the like may be introduced consecutively byrestriction enzyme cleavage of an appropriate replication system andinsertion of the particular construct or fragment into the availablesite. After ligation and cloning the vector may be isolated for furthermanipulation. All of these techniques are amply exemplified in theliterature and find particular exemplification in Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1982.

Once the vector is complete, the construct may now be introduced intoplant cells. Techniques for transforming plant cells includemicroinjection, direct DNA uptake using polyethylene glycol,electroporation, viral infection, and transformation with Agrobacterium.In accordance with the subject invention, a functional method has beendeveloped for transformation of Brassica cells employing Agrobacterium.This technique provides a methodology for the transformation of plantspecies with foreign genes in an efficient manner, so as to provide arapid technique for transforming plant cells and regeneration of plantsin an efficient reproducible manner.

Target Tissues

Although the prior art teaches that Brassica can be regenerated fromnumerous plant tissues, hypocotyls have been found to provide thegreatest efficiency of transformation. Other plant parts such as leafexplants, may be used in conjunction with the subject selection andregeneration medium. However, use of hypocotyl tissue represents apreferred embodiment of the present invention.

Sterile seeds are preferably employed as a source for Brassicahypocotyls. Surface sterilized leaf pieces from 3-week-old plants orsterile grown hypocotyls of Brassica napus cv Westar both regeneratereadily. The cut surfaces of these explants provides an idealAgrobacterium target.

Any Brassica species can be used in the practice of the presentinvention such as B. napus (rape seed and rutabaga), B. oleraceae(cabbage, broccoli, brussel sprouts and other oleracea varieties), B.juncea (indian mustard), B. campestris (turnip rape), and the like.

Use of Feeder Cells During Transformation

Feeder cells can be used in the transformation process. The cells of thefeeder plates act as a nurse culture for the Brassica explant as well asenhancing the efficiency of the transformation rate. In general tobaccofeeder cells are used for ease of manipulation. Other feeder cells couldbe employed, particularly Brassica feeder cells in the form of a finesuspension.

The feeder plates are prepared by employing a plant suspension culture(e.g., Nicotiana cells grown in Murashige minimal organic medium (FlowLab.) supplemented with 0.2 mg/l 2,4-dichlorophenoxyacetic acid and 0.1mg/l kinetin) on a soft agar medium, generally having from about 0.5 to1% agar and containing an appropriate growth medium, such as Gamborg,Miller and Ojima salts (B5 salts) ref. Gamborg et al., Exp. Cell. Res.(1968) 50:151-158, a carbon source, e.g., sucrose (3%), and appropriateamounts of growth substances, i.e., auxins, such as2,4-dichlorophenoxyacetic acid (2,4-D), kinetin and vitamins (such asthiamine), with the medium appropriately buffered in the range from 5 to6, preferably about 5.5. 2,4-D and kinetin concentrations are 1 mg/l.The final concentration of the vitamins and supplements is as follows:Inositol (100 mg/l), Nicotinic acid (1mg/l), Pyridoxine HCl (1 mg/l),Thiamine HCl (10 mg/l). Desirably, the feeder plates are prepared priorto being used, usually 24-48 hours before being used.

The feeder plates are covered with a porous cover to prevent the feedercells from coming into contact with the Brassica leaf or shoot explants.This porous cover allows the explants to be bathed in conditionedmedium. This can be readily achieved employing a sterile filter paperdisk. The explants are then allowed to preincubate, followed by transferto a broth culture of the Agrobacterium strain containing theconstruction for integration and having the genetic capability fortransfer of the construct into the plant cells. Generally, the number ofbacteria is from about 10⁷ to 10⁹ /ml (final concentration). The contacttime with the bacteria in the bacterial broth culture, e.g., MG/L (sameas LBMG; see Garfinkel et al., J. Bacteriol. (1980) 144:732-743), ispreferably about 30 minutes to 1 hour, although longer or shorter timesmay be used. The explants are then transferred from the bacterial broth,excess surface liquid is removed, and the sections are returned to thefeeder plates. Bacterial cocultivation on the feeder plates is for atleast 12 hours and not more than 3 days, averaging 1-2 days.

Selection and Regeneration Procedures

It has been found that use of a low-carbon-source medium during theregeneration procedure following co-cultivation of the Brassica cellswith the transforming bacteria results in enhanced recovery andregeneration. Use of a low-carbon-source medium is believed to operateby forcing the cultured tissues to become dependent on other sources ofenergy, possibly photosynthesis. A low-carbon-source medium is one thatcontains less than 2% by weight sucrose or the equivalent of a 2% byweight sucrose solution in caloric value. Other carbon sources (e.g.,mono- or di-saccharides) may give a similar effect provided that theyprovide the same caloric value. A typical salt and vitamin mixture isemployed in conjunction with cytokinins for regeneration media.

After the 1-2 day co-cultivation with the bacteria described above, theexplants are typically transferred to a Brassica callus media (whichpreferably contains B5 salts and vitamins, 1mg/ 2,4-D and kinetin, 3%sucrose). Cytokinins may be excluded and their absence enhancesregeneration frequency. The callus forming medium contains abacteriocide, e.g., carbenicillin (500 mg/L) and a selective agent maybe applied. For example, with the kanamycin resistance gene (neomycinphosphotransferase, NPTII) as the selective marker, kanamycin at aconcentration of from about 10 to 200 mg/l may be included in themedium. Typical concentrations for selection are 10-50 mg/l althoughsome transformants may be resistant to 200 mg/l kanamycin. The tissue isgrown upon this medium for a period of 1 to 3 weeks, preferably about 7days.

After this time the callusing explants are transferred to Brassicaregeneration medium. This medium contains Gamborg. Miller and Ojima B5salts and vitamins as described below, 1% sucrose, 3-benzyladenine (3mg/l), zeatin (1 mg/l) 0.6% purified agar (Phytagar, Gibco), andcarbenicillin at 500 mg/l. At this stage a selective agent may beapplied. Shoot formation begins in about 3-6 weeks depending ontreatment and co-cultivation conditions. Kanamycin-resistant callus,which is also potentially regenerable, grows in a similar time. Bothregenerants and transformed callus are removed and regularly (everyother week) transferred to fresh B5 medium containing the othercomponents described immediately above. Failure to perform regulartransfers results in loss of transformants and depression of apparenttransformation rate.

The Brassica transformation and regeneration system described above hasbeen found to be rapid and efficient. A sufficient percentage of theco-cultivated explants are transformed in order to provide an economicsystem for transforming Brassica.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

E. coli strain MM294 (Hanahan, J. Mol. Biol. (1983) 116:557-580) wasused as the host for binary vectors containing the pRK290 type replicon.Strain K12 was generated by transforming pTiA6 into strain A114 (NT1)(Nester and Kosuge, Ann. Rev. Microbiol. (1981) 35:531; Hoekema et al.,Nature (1983) 303:179). Agrobacterium strain A281 was generated byconjugating wild-type Bo542, with strain A136 (D. Sciaky, et al.,Plasmid (1978) 1:238-253). Agrobacterium strain EHA101 is described byHood et al. J. of Bacteriology (1986) 168: 1291-130)

Levels of antibiotics used with E. coli in mg/l were 30 for kanamycin,50 for chloramphenicol, 300 for penicillin, 10 for tetracycline, and 20for gentamicin. Unless otherwise indicated, levels of antibiotics usedwith Agrobacterium in mg/l were 100 for kanamycin or gentamicin and 50for carbenicillin or chloramphenicol.

Laboratory Procedures

Restriction enzymes and T4 ligase were obtained from commercial sourcesand used according to manufacturers' recommendations. Standard methodsof cloning and molecular analysis were performed as described inManiatis et al., supra.

Designations of media used in these procedures are as follows:

B5 medium means that described by Gamborg, Miller and Ojima, Exp. CellResearch (1968) 50:151-158.

    ______________________________________                                        B5 Salts are:          mg/l                                                   ______________________________________                                        Ammonium sulfate       134.0                                                  Boric acid             3.0                                                    Calcium chloride       150.0                                                  Cobaltous chloride     0.025                                                  Cupric sulfate         0.025                                                  Ferrous sulfate        27.8                                                   Magnesium sulfate      250.0                                                  Manganese sulfate      10.0                                                   Potassium iodide       0.75                                                   Potassium nitrate      2500.0                                                 Sodium ethylenediamine tetraacetate                                                                  37.3                                                   Sodium molybdate       0.25                                                   Sodium dihydrogen phosphate                                                                          150.0                                                  Zinc sulfate           2.0                                                    ______________________________________                                    

Unless otherwise stated B5 medium contains 3% sucrose weight/volume.

    ______________________________________                                        B5 vitamins and supplements are:                                                                    mg/l                                                    ______________________________________                                        myo-Inositol          100.0                                                   Nicotinic acid        1.0                                                     Pyridoxine HCl        1.0                                                     Thiamine HCl          10.0                                                    ______________________________________                                    

The designation B5 0/1/1 means B5 medium, 3% sucrose, 1 mg/l 2,4-D, 1mg/l kinetin and the designation B5 0/1/0 is the same except the kinetinis not included. The growth substance concentrations are denoted by the/ (slash) notation, with the concentration of 3 growth substances inmg/l being denoted. In order these are indole-3-aceticacid/2,4-D/kinetin.

The designation B5BZ 1% means B5 salts, vitamins and supplements plus 1%w/v sucrose, 3 mg/l benzyl adenine and 1 mg/l zeatin. B5BZ 1% is usedhere as regeneration and selection medium for kanamycin resistance ofBrassica tissue.

EXAMPLE I Construction of pCGN587

The BglII-SmaI fragment of Tn5 containing the entire structural gene forAPHII (Jorgensen et al., Mol. Gen. (1979) 177:65) was cloned into pUC8(Vieira and Messing, Gene (1982) 19:259), converting the fragment into aHindIII-EcoRI fragment, since there is an EcoRI site immediatelyadjacent to the SmaI site. The PstI-EcoRI fragment containing the 3'portion of the APHII gene was then combined with anEcoRI-BamHI-SalI-PstI linker into the EcoRI site of pUC7 (pCGN546W).Since this construct does not confer kanamycin resistance, kanamycinresistance was obtained by inserting the BglII-PstI fragment of theAPHII gene into the BamHI-PstI site (pCGN546X). This procedurereassembles the APHII gene, so that EcoRI sites flank the gene. An ATGcodon was upstream from and out of reading frame with the ATG initiationcodon of APHII. The undesired ATG was avoided by inserting aSauIIIA-PstI fragment from the 5'-end of APHII, which fragment lacks thesuperfluous ATG, into the BamHI-PstI site of pCGN546W to provide plasmidpCGN550.

The EcoRI fragment containing the APHII gene (1ATG) was then cloned intothe unique EcoRI site of pCGN451, which contains an octopine synthasecassette for expression to provide pCGN552(1ATG).

Plasmid pCGN451 includes an octopine cassette which contains about 1556bp of the of the 5' non-coding region fused via an EcoRI linker to the3' non-coding region of the octopine synthase gene of pTiA6. The pTicoordinates are 11,207 to 12,823 for the 3' region and 13,643 to 15,208for the 5' region as defined by Barker et al., Plant Mol. Biol. (1983)2:325.

The 5' fragment was obtained as follows. A small subcloned fragmentcontaining the 5' end of the coding region, as a BamHI-EcoRI fragmentwas cloned in pBR322 as plasmid pCGN407. The BamHI-EcoRI fragment has anXmnI site in the coding region, while pBR322 has two XmnI sites. pCGN407was digested with XmnI, resected with Ba131 nuclease and EcoRI linkersadded to the fragments. After EcoRI and BamHI digestion, the fragmentswere size fractionated, the fractions cloned and sequenced. In one case,the entire coding region and 10 bp of the 5' non-translated sequenceshad been removed leaving the 5' non-transcribed region, the mRNA capsite and 16 bp of the 5' non-translated region (to a BamHI site) intact.This small fragment was obtained by size fractionation on a 7%acrylamide gel and fragments approximately 130 bp long eluted. This sizefractionated DNA was ligated into M13mp9 and several clones sequencedand the sequence compared to the known sequence of the octopine synthasegene. The M13 construct was designated pI4, which plasmid was digestedwith BamHI and EcoRI to provide the small fragment which was ligated toa XhoI to BamHI fragment containing upstream 5' sequences from pTiA6(Garfinkel and Nester, J. Bacteriol. (1980) 144:732) and to an EcoRI toXhoI fragment containing the 3' sequences. The resulting XhoI fragmentwas cloned into the XhoI site of a pUC8 derivative, designated pCGN426.This plasmid differs from pUC8 by having the sole EcoRI site filled inwith DNA polymerase I, and having lost the PstI and HindIII sites bynuclease contamination of HincII restriction endonuclease, when a XhoIlinker was inserted into the unique HincII site of pUC8. The resultingplasmid pCGN451 has a single EcoRI site for the insertion of proteincoding sequences between the 5' non-coding region (which contains 1,550bp of 5' non-transcribed sequences including the right border of theT-DNA, the mRNA cap site and 16 bp of 5' non-translated sequence) andthe 3' region (which contains 267 bp of the coding region, the stopcodon, 196 bp of 3' non-translated DNA, the polyA site and 1,153 bp of3') non-transcribed sequence).

The resulting plasmid pCGN451 having the ocs 5' and the ocs 3' in theproper orientation was digested with EcoRI and the EcoRI fragment frompCGN451 containing the intact kanamycin resistance gene was insertedinto the EcoRI site to provide pCGN552 having the kanamycin resistancegene in the proper orientation.

This ocs/KAN gene was used to provide a selectable marker for thetrans-type binary vector pCGN587.

The 5' portion of the engineered octopine synthase promoter cassetteconsists of TiA6 DNA from the XhoI fragment at bp 15208-13644 (Barker'snumbering as in Barker et al. supra), which also contains the T-DNAboundary sequence (border) implicated in T-DNA transfer. In the plasmidpCGN587, the ocs/KAN gene from pCGN552 provides a selectable marker aswell as the right border. The left boundary region was recloned from theHindIII-EcoI fragment as a KpnI-EcoRI fragment in pCGN565 to providepCGN580. pCGN565 is a cloning vector based on pUC8-Cm, but containingpUC18 linkers. pCGN580 was linearized with BamHI and used to replace thesmaller BglII fragment of pVCK102 (Knauf and Nester, Plasmid (1982)8:45), creating pCGN585. By replacing the smaller SalI fragment ofpCGN585 with the XhoI fragment from pCGN552 containing the ocs/KAN gene,pCGN587 was obtained.

Construction of pCGN200

To construct pCGN200, a plasmid containing a full-length promoter fromCaMV (35S) and kanamycin gene as a selectable marker, pCGN167, wasrecombined into a binary vector, pCGN587.

To construct pCGN167, the AluI fragment of CaMV (bp 7144-7735) (R.Gardner et al., Nucl. Acid Res. (1981) 9.2871-2888) was obtained bydigestion with AluI and cloned into the HincII site of M13mp7 (Vieira,et al., Gene (1982) 19:259) to create C614. An EcoRI digest of C614produced the EcoRI fragment from C614 containing the 35S promoter whichwas cloned into the EcoRI site of pUC8.(Vieira et al., Gene (1982)19:259) to produce pCGN146.

To trim the promoter region, the BglII site (bp 7670) was treated withBglII and Ba131 and subsequently a BglII linker was attached to theBa131 treated DNA to produce pCGN147.

pCGN148a containing a promoter region, selectable marker (KAN with 2ATG's) and 3' region, was prepared by digesting pCGN528 (see below) withBglII and inserting the BamHI-BglII promoter fragment from pCGN147. Thisfragment was cloned into the BglII site of pCGN528 so that the BglIIsite was proximal to the kanamycin gene of pCGN528.

The shuttle vector used for this construct, pCGN528, was made asfollows. pCGN525 was made by digesting a plasmid containing Tn5 whichharbors a kanamycin gene (Jorgenson et al., Mol. Gen. (1979) 177:65)with HindIII-BamHI and inserting the HindIII-BamHI fragment containingthe kanamycin gene into the HindIII-BamHI sites in the tetracycline geneof pACYC184 (Chang & Cohen, J. Bacteriol. (1978) 134:1141-1156). pCGN526was made by inserting the BamHI fragment 19 of pTiA6 (Thomashaw et al.,Cell (1980) 19:729-739) into the BamHI site of pCGN525. ,pCGN528 wasobtained by deleting the small XhoI fragment from pCGN526 by digestingwith XhoI and religating.

pCGN149a was made by cloning the BamHI-kanamycin gene fragment frompMB9KanXXI into the BamHI site of pCGN148a.

pMB9KanXXI is a pUC4K variant (Vieira & Messing, Gene (1982) 19:259-268)which has the XhoI site missing but contains a functional kanamycin genefrom Tn903 to allow for efficient selection in Agrobacterium.

pCGN149a was digested with BglII and SphI. This small BglII-SphIfragment of pCGN149a was replaced with the BamHI-SphI fragment from MI(see below) isolated by digestion with BamHI and SphI. This producespCGN167, a construct containing a full length CaMV promoter,1ATG-kanamycin gene, a 3' end and the bacterial Tn903-type kanamycingene. MI is an EcoRI fragment from pCGN546X (see construction ofpCGN587) and was cloned into the EcoRI cloning site of M13mp9 in such away that the PstI site in the 1ATG-kanamycin gene was proximal to thepolylinker region of M13mp9.

pCGN200 was made by transforming an E. coli strain C2110 (polA1)containing the binary vector pCGN587 with pCGN167. pCGN167 recombined invivo to make pCGN200. There are two regions of direct DNA homology bywhich recombination could have occurred. In this case recombination tookplace between the pUC origin of replication regions carried by pCGN167and pCGN587. Recombinants were selected by kanamycin resistance(deFromard et al., Biotechnology, May 1983, pp. 262-267).

pCGN200 was introduced into Agrobacterium tumefaciens A281 and K12 bymating. Bacterial matings were performed using two E. coli strains andone Agrobacterium strain. One E. coli strain (MM294) harbored pRK2073which provided mobilization functions, and the other strain (C2110)carried the plasmid with a kanamycin resistance marker to be transferredinto Agrobacterium. The two E. coli strains were grown overnight at 37°C. with shaking in LB broth. The Agrobacterium strain was grownovernight at 28° C. in MG/L broth. Fifty microliters of each of thethree strains were mixed on a nitrocellulose filter and placed on anMG/L plate. The plate was incubated at 28° C. for 3 days. The mixturewas then streaked onto an AB minimal medium (D. M. Glover, DNA CloningVolume II (1985) p. 78) supplemented with 100 μg/ml kanamycin and 100μg/ml streptomycin and incubated at 28° C. for two days. Streptomycinwas included to kill the two E. coli strains. Single colonies werepicked and purified by two more streakings on the above medium.

Construction of strain K61

The Ti plasmid pTiA6 was isolated from Agrobacterium strain A348(Garfinkel et al., Cell (1981) 27:143-153) and used to transformAgrobacterium strain A114 (also designated NT1) (Currier and Nester, J.Bacteriol. (1976) 126:157-165). Octopine catabolism was selected for onBTB media (Hooykaas et al., J. Gen. Microbiol. (1979) 110:99-109). Thisstrain was named K12.

The left T-DNA border region of pTiA6 (bp 602 to 2212 by the numberingsystem of Barker et al., supra) was cloned as a HindIII to SmaI fragmentin the phage vector M13mp9 (pCGN501). The M13mp9 linker architectureprovides the fragment as a HindIII to EcoRI fragment. The right T-DNAborder region of pTiA6 (bp 13362 to 15208, Barker et al., supra) wassubcloned as a EcoRI to XhoI fragment into M13mp9 cut with enzymes EcoRIand SalI (pCGN502). This piece could then be excised as a EcoRI toHindIII fragment. The plasmids pCGN501 and pCGN502 were digested withHindIII and EcoRI and ligated to pUC8 DNA previously cut with justHindIII. Selection of while penicillin resistant colonies yielded anisolate containing the pUC8 vector with a 3.5 kbp HindIII fragmentcontaining the left and right T-DNA borders of pTiA6 in the naturalorientation (pCGN503). This 3.5 kpb HindIII fragment containing T-DNAborders was then transferred into the HindIII site of pVK102 (pCGN506).The wide host range cloning vector pVK102 (also designated pVCK102) hasbeen described (Knauf and Nester, Plasmid (1982) 8:45-54). Theorientation of the border fragment in pCGN506 relative to the pVK102vector is the left T-DNA border region proximal to the tetracyclineresistance locus. pCGN506 has a unique EcoRI site and a unique BamHIsite in between the left and right border regions, i.e., such that anyinserts would be oriented in the natural orientation. The plasmidpCGN567 was constructed by ligating the BamHI fragment of pUC4K bearinga kanamycin resistance determinant into the BamHI site of pCGN506. Thus,pCGN567 codes for both tetracycline and kanamycin resistance.

The plasmid pCGN567 was mated into Agrobacterium strain K12 by thetripartite method (Ditta et al., Proc. Natl. Acad. Sci. USA (1981)77:7347-7351) using kanamycin selection for transconjugant Agrobacteriaon minimal media. E. coli bearing the plasmid pPH1J1 (Garfinkel et. al.,Cell (1981) 27:143-153) was then mated with strain K12 (pCGN567) andtransconjugant Agrobacteria were selected on minimal media containingboth kanamycin and gentamicin. Since pPH1J1 and pCGN567 are incompatibleplasmids, it was expected that a double recombination event between twodirect regions of homology with the Ti-plasmid would result in theexchange of the kanamycin resistance locus for all of the oncogenic geneloci between the border regions (for explanation of method, seeGarfinkel et al., Cell (1981) 27:143-153,). However, this did not occur.Kanamycin and gentamicin resistant Agrobacterium resulting from theintroduction of pPH1JI into K12 (pCGN567) grew very slowly suggestingboth pCGN567 and pPH1JI were present in an unstable situation resultingin the apparent slow growth of a colony as individual bacteria tended toshed one of the two plasmids due to incompatibility problems. Byalternating growth on kanamycin-containing media andgentamicin-containing liquid media in overnight cultures, an isolatewith the expected growth rates was identified. Plasmid DNA was isolatedfrom that strain and used to transform strain A114, selecting forkanamycin resistance and toothpicking for gentamicin sensitivity. Theresulting strain of Agrobacterium, K61, therefore lacked pPH1JI.Restriction enzyme analysis of the plasmid in K61 (pTiK61) using SmaI,HpaI, EcoRI, and SalI revealed a Ti-plasmid lacking sequencecorresponding to kbp 173.30 to 181 and 0.0 to 53.35 of pTiA6 (using thenumbering system of Knauf and Nester, Plasmid (1982) 8:45-54). Thisexplained the inability of strain K61 to utilize octopine, a traitnormally encoded by pTiA6. Thus, pTiK61 represents a spontaneousdeletion of pTiA6 in which the left border region is still present butthe oncogenes, right T-DNA border, the T_(R) -DNA region and other lociincluding octopine catabolism are deleted. Regions encoding the virregion of pTiA6, namely coordinates 111 through 168 (using the numberingsystem of Knauf and Nester, supra) are intact in pTiK61 which representsa disarmed Ti-plasmid lacking oncogenes or functional T-DNA butcontaining vir genes necessary to accomplish T-DNA transfer from binaryT-DNA vectors.

Construction of pCGN767

A genomic library was constructed in the λ vector EMBL4 (Fischauf etal., J. Mol. Biol. (1983) 170:827-842) from B. napus DNA digestedpartially with SauIIIA. Two unique napin genomic clones, designatedλBnNa and λBnNb, were isolated when 4×10⁵ recombinant phage werescreened by plaque hybridization with a nick-translated pN1 napin cDNAprobe (Crouch et al., J. Mol. Appl. Gen. (1983) 2:273-283).

The napin genomic clones were analyzed by restriction nuclease mappingand Southern blot hybridizations. Each phage contains just one napingene, and only the napin gene region hybridizes to cDNA made from embryoRNA. The 3.3 kb EcoRI fragment containing the λBnNa napin gene wassubcloned in pUC8 (Veiera and Messing, 1982) and designated pgNa.

An approximate 320 bp SalI fragment was cloned into the XhoI site ofpgNa to create pCGN714 placing a bacterial DNA sequence as a "tag" afterthe stop codon of the napin coding region in pgNa. In this case, thebacterial DNA sequence consisted of a SalI restriction fragmentcontaining the coding region of a dihydrofolate reductase (DHFR) gene.The EcoRI fragment in pgNa containing the napin gene with 300 bp ofpromoter and approximately 2100 bp following the napin coding region isabout 3.6 kb in pCGN714.

The HindIII-EcoRI set of linkers in pUC18 (Yanisch-Perron et al., Gene(1985) 33:103) were transferred into pUC12Cm (Keith J. Buckley, Ph. D.Thesis, USCD, 1985) to create pCGN565 which is basically a pUC repliconlinked to chloramphenicol resistance and the blue-white cloning systemof pUC12. The EcoRI fragment of pCGN714 containing the tagged napin genewas transferred to the EcoRI site of pCGN565 to get pCGN723 which codesfor chloramphenicol resistance rather than the penicillin resistance ofpCGN714. The HindIII-EcoRI linkers of pUC8Cm (Keith J. Buckley, Ph. D.,Thesis, UCSD, 1985) were transferred into HindIII-EcoRI cut pEMBL19(Dente et al. Nucl. Acids. Res. (1983) 11:1645) making pCGN730a. Unlikethe parent pEMBL19, pCGN730a lacks any SstI sites. The EcoRI fragment ofpCGN723 containing the tagged napin gene was transferred to the EcoRIsite of pCGN730a. The SstI site in the napin gene was then unique in theresulting plasmid, pCGN735.

Two 27mer oligdnucleotides were synthesized on an Applied Biosystems DNAsynthesizer machine. #A consisted of the sequence:

C-C-T-G-A-T-G-A-T-G-A-T-G-A-T-G-A-T-G-C-T-G-C-A-G-C-T in the order of 5'to 3'. #B consisted of the sequence:

G-C-A-G-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-G-G-A-G-C-T-

These two oligonucleotides are partially complementary so that byannealing, they leave 3' sticky ends compatible with cloning in SstIsites. In the desired orientation, a single insert adds coding for anadditional 9 amino acids, 5 of which are methionine residues. Thesynthetic dsDNA created by annealing oligonucleotides #A and #B wascloned into the SstI site of pCGN735. Since the oligonucleotides werenot phosphorylated, the insertion of only one element was likely eventhough the insert was in excess to pCGN735 SstI ends. Restriction enzymeanalysis of that plasmid, pCGN757, indicated that an insert was present(this and the orientation were later verified by DNA sequencing). TheEcoRI fragment of pCGN757 was transferred to pCGN565 so that plasmidpCGN757c, coded for chloramphenicol resistance rather than thepenicillin resistance of pCGN757,

pCGN549 was made by cloning the EcoRI (coding for gentamicin resistance)of pPH1Jl (Hirsch and Beringer, Plasmid (1984) 12:139) into EcoRI andPstI cut pUC9. pCGN594 was made by cutting pCGN587 with HindIII andBglII and combining with pCGN549 cut with HindIII and BamHI. Thisreplaced the pUC replicon and chloramphenicol marker of pCGN587 with abacterial gentamicin marker. pCGN739 was made by replacing theHindIII-BamHI fragment of pCGN594 with the HindIII-BamHI linkers ofpUC18. This effectively replaced the eukaryotic selectable marker ofpCGN594 with a series of unique cloning sites for insertion of othertypes of selectable markers. pCGN763 was made by the transfer of theHindIII-BamHI fragment of pCGN976 (obtained by insertion of aHindIII-BamHI fragment from pCGN167 into HindIII-BamHI digested pUC19 tointroduce the 35S promoter, kanamycin resistance and tml 3, region) intoHindIII-BamHI cut pCGN739. pCGN757c was linearized with HindIII andcloned into the HindIII site of pCGN763 to create the binary vectorpCGN767 which includes a bacterial gentamicin resistance marker, achimeric eukaryotic kanamycin resistance gene (with CaMV 35S promoterand pTiA6 T-DNA tml transcription termination signals), and the tagged,engineered napin gene between pTiA6 T-DNA borders.

Example II Brassica Transformation

Explants from soil-grown seedlings of Brassica napus cv Westar(Agriculture Canada, Saskatoon, Canada) were used as primary targetmaterial. Plants were grown 3-4 weeks in a 16-8 hour light-dark cycle220 μEm⁻² S⁻¹ at 24° C. Partially expanded secondary leaves wereexcised, surface sterilized for 15 min in 1% sodium hypochlorite, andwashed four times with sterile water.

Leaf discs 4 mm in diameter were cut from the sterile leaves using acork borer. These discs were pre-incubated for 24 hours at 24° C. indarkness on a B5 medium (KC Biologicals) containing 1 mg/l 2,4-D and 1mg/l kinetin solidified using 0.6% purified agar (Phytagar) (B5 0/1/1).

Agrobacterium tumefaciens (strain A281x200) was prepared overnight inMG/L broth by incubating a single colony of Agrobacterium tumefaciens.Bacteria were harvested at time periods of 16-36 hours. Dilutions ofbacteria to concentrations of 10⁶ -10⁷ bacteria per ml were prepared inB5 0/1/1 liquid medium. Leaf explants were inoculated with bacteria bydipping into the Agrobacterium suspension and then lightly blotting themon sterile paper towels. Inoculated leaf discs were then transferred toPetri plates of B5 0/1/1 medium with 0.6% Difco Phytagar, at a densityof 20 discs per plate.

The co-incubation of bacteria and leaf discs took place for periods from12 to 48 hours. After this co-culture step, the discs were washed inliquid B5 0/1/1 medium and transferred to Petri plates containing B50/1/1 and 500 mg/l carbenicillin, 0.6% Difco Phytagar. These explantswere cultured in light (50 μEm⁻² s⁻¹) on this medium for 7-10 days untilcallus formation was evident. At this time the explants were transferredto a second medium optimized for regeneration in Brassica napus cvWestar. This contained B5 salts and vitamins, 3 mg/l benzyl adenine, 1mg/l zeatin, and 1% sucrose. It was supplemented with 500 mg/lcarbenicillin and 50 mg/l kanamycin sulfate. This medium was solidifiedusing 0.7% Phytagar. Under lighted conditions (16-8 light-dark cycles at24° C., 100 μEm⁻² s⁻¹) the tissue began to develop green callus. Undernon-selective conditions without kanamycin sulfate) numerous shoots formon this medium which can be propagated and rooted. Under selectiveconditions green callus and shoot formation is evident, but greatlyreduced.

Under these selective conditions successful transformation eventsleading to kanamycin-resistant material will grow and may be scored forfrequency. To assure that the primary selection pressure is forkanamycin resistance and not nutrient scavenging or insensitivity toinhibitors released from dying tissue, the explants were re-plated onthe identical medium every 7-10 days.

Shoots and callus growing on the kanamycin-containing medium may betested for the expression of the neomycin phosphotransferase gene usingan assay described by Reiss et al., Gene (1984) 30:211-218. This employspolyacrylamide gel electrophoresis to separate the enzyme frombackground proteins. Enzyme activity is detected in situ by ATP mediatedphosphorylation of kanamycin using γ-³² P-labelled ATP. The product ofthe reaction is blotted onto P81 ion exchange paper which is thentreated (45 min at 65° C.) with Proteinase K (1 mg/l Sigma Chemicals) in1% sodium dodecyl sulfate. This treatment removes much of the backgroundradioactivity on the paper associated with ³² P-labelled proteins. Thephosphorylated kanamycin remains intact during the treatment. Thisproduct is then detected by autoradiography and may be quantified byscintillation counting. An example of such an assay performed ontransformed, kanamycin resistant Brassica napus tissue is shown inFIG. 1. The phenotype of the transformant is kanamycin resistance. Thelevel of resistance in a Brassica A281×200 transformant is shown in FIG.2.

EXAMPLE III

Transformation may be conducted using Agrobacterium tumefaciens in thepresence of a feeder cell layer. This may be advantageous both to helpthe Agrobacterium-treated tissue recover and for stimulation oftransformation activity. In this embodiment the tissues are prepared asdescribed in Example II, but then are transferred, after dipping intoAgrobacterium and blotting, onto Petri plates containing feeder cells ofNicotiana tabacum (tobacco) suspension cells. The feeder plates areprepared by pipetting 1.0 ml of a stationary phase tobacco suspensionculture onto B5 medium containing 1 mg/l of both 2,4-D and kinetin withvitamins as described above. The medium is solidified using 0.6% agar.The feeder plates are produced 24-48 hours prior to use, and the excisedBrassica tissue may be pre-incubated on the feeder plate by placing asterile Whatman 3 mm filter paper on top of the feeder layer andarranging the excised Brassica tissue on this 24 hours prior toAgrobacterium treatment.

After dipping in Agrobacterium tumefaciens (A281×200 or similar strain)the Brassica explants are returned to the feeder plates for a further24-48 hours. After this time they are transferred to B5 mediumcontaining 1 mg/l kinetin, 1 mg/l, 2,4-D and 500 mg/l carbenicillin inagarized medium (0.6%). All other steps are identical to those describedin Example II.

EXAMPLE IV

This transformation method can also be applied effectively to hypocotylexplants rather than leaf explants. All procedures for transformation ofthe hypocotyl explants are identical to those described above for leafdiscs; however, the preparation of hypocotyl material differs.

Seeds of Brassica napus cv Westar were surface sterilized in a 1% sodiumhypochlorite solution containing 200 μl of "Tween 20" surfactant per 500ml of sterilant solution. After 20 minutes soaking in the sterilant theseeds were washed (4 times) with sterile distilled water and planted insterile plastic boxes 7 cm wide, 7 cm long, and 10 cm high (Magenta)containing 50 ml of 1/10 concentrated B5 medium (Gamborg, Miller andOjima, Experimental Cell. Res. (1968) 50:151-158) containing no growthsubstances and solidified with 0.6% agar. The seeds germinated and weregrown at 23°-25° C. in a 16-8 hour light-dark cycle with light intensityapproximately 100 μEm⁻² s⁻¹. After 5 days the seedlings were taken understerile conditions and the hypocotyls excised and cut into pieces ofabout 4 mm in length. These hypocotyl segments were then treated withall the same procedures applied to leaf disc explants in Example II.

EXAMPLE V

Seeds of Brassica napus cv. Westar were soaked in 95% ethanol for 4minutes. They were sterilized in 1% solution of sodium hypochlorite with50 μl of "Tween 20" surfactant per 100 ml sterilant solution. Aftersoaking for 45 minutes, seeds were rinsed 4 times with sterile distilledwater. They were planted in sterile plastic boxes 7 cm wide, 7 cm long,and 10 cm high (Magenta) containing 50 ml of 1/10th concentration of MS(Murashige minimal organics medium, Gibco) with added pyridoxine (50μg/l), nicotinic acid (50 ug/l), glycine (200 ug/l) and solidified with0.6% agar. The seeds germinated and were grown at 22° C. in a 16-8 hourlight-dark cycle with light intensity approximately 65 μEm⁻² s⁻¹. After5 days the seedlings were taken under sterile conditions and thehypocotyls excised and cut into pieces of about 4 mm in length. Thehypocotyl segments were placed on a feeder plate described in ExampleIII or without the feeder layer on top of a filter paper on thesolidified B5 0/1/1 medium. This was done 24 hours prior toAgrobacterium treatment.

Agrobacterium tumefaciens (strains A281×767 and EHA101×767) wereprepared by incubating a single colony of Agrobacterium in MG/L broth at30° C. Bacteria were harvested 16 hours later and dilutions of 10⁸bacteria per ml were prepared in MG/L broth. Hypocotyl segments wereinoculated with bacteria by placing in Agrobacterium suspension andallowed to sit for 30-60 minutes, then removed and transferred to Petriplates containing B5 0/1/1 medium described above. The plates wereincubated in low light at 22° C. The co-incubation of bacteria with thehypocotyl segments took place for 24-48 hours. The hypocotyl segmentswere removed and placed on B5 0/1/1 containing 500 mg/l carbenicillin(kanamycin sulfate at 10, 25, or 50 mg/l was sometimes added at thistime) for 7 days in continuous light (approximately 65 μEm⁻² s⁻¹) at 22°C. They were transferred to B5 medium with 3 mg/l BAP and 1 mg/l zeatinas described in Example III. This was supplemented with 500 mg/lcarbenicillin, 10, 25, or 50 mg/l kanamycin sulfate, and solidified with0.6% Phytagar (Gibco). Thereafter explants were transferred to freshmedium every 2 weeks.

After 1 month green shoots developed from green calli which wereselected on media containing kanamycin. Shoots continued to developfor.3 months. The shoots were cut from the calli when they were at least1 cm high and placed on B5 medium with 1% sucrose, no added growthsubstances, 300 mg/l carbenicillin, and solidified with 0.6% phytagar.The shoots continued to growth and several leaves were removed to testfor neomycin phosphotransfersase II (NPTII) activity. Shoots which werepositive for NPTII activity were placed in Magenta boxes containing B5medium with 1% sucrose, 2 mg/l indolebutyric acid, 200 mg/lcarbenicillin, and solidified with 0.6% Phytagar. After a few weeks theshoots developed roots and were transferred to soil.. The plants weregrown in a growth chamber at 22° C. in a 16-8 hours light-dark cyclewith light intensity 220 μEm⁻² s⁻¹ and after several weeks weretransferred to the greenhouse.

Leaves were harvested, frozen in liquid nitrogen and DNA extracted(Dellaporta et al., Pl. Molec. Biol. Reporter (1983) 1:19-21). Southernanalysis (Maniatis et al., "Molecular Cloning", Cold Spring HarborPress) confirmed proper integration of the T-DNA.

Using the method described above 2% of the hypocotyl segments producedshoots which were positive for NPTII activity.

    ______________________________________                                        Frequency of hypocotyl explants producing NPTII                               positive shoots                                                                         Agrobacterium Strain                                                Date      EHA101pCGN767  A281 pCGN767                                         ______________________________________                                        1         1/59 2%        1/59 2%                                              2         1/57 2%                                                             3         1/59 2%        1/54 2%                                                        1/62 2%        1/60 2%                                                        1.64 2%        1/59 2%                                                        2/58 3%        1/60 2%                                                        1/58 2%        1/59 2%                                                        2/61 3%        1/59 2%                                              ______________________________________                                    

Transgenic plants have been also obtained from hypocotyl segments of B.napus cultivars Westar, Viking and Bridger cocultivated withAgrobacterium strains EHA101 and K61 containing other constructs withother plant genes. The system is repeatable with different Agrobacteriumstrains, constructs, and Brassica geneotypes.

EXAMPLE VI

The shoot regeneration frequency from hypocotyl segments was increasedat least two-fold by removing kinetin from the cocultivation andcallusing medium (B5 0/1/1 to B5 0/1/0).

    ______________________________________                                        Frequency of hypocotyl explants producing at least one                        shoot                                                                         B. napus      Callusing medium                                                Date    cultivar  B5 0/1/0     B5 0/1/1                                       ______________________________________                                        1       Westar    13/22    59%    3/24  12%                                   2       Westar    12/20    60%    2/28   7%                                   3       Westar    17/23    74%    3/24  12%                                   4       Westar    26/29    90%   12/28  43%                                   5       Viking     95/100  95%    53/102                                                                              52%                                   6       Bridger    58/101  57%   13/77  17%                                   ______________________________________                                         This change provides an increased number of transformed shoots recovered     from hypocotyl explants which were inoculated with Agrobacterium.

The above results demonstrate that Brassica species can be transformedefficiently, whereby foreign genes may be integrated into the plantgenome and expressed, providing novel phenotypic properties. Thus,Brassica species can be transformed and are shown to be capable ofutilizing genes where the transformed cells may be regenerated intoplants which provide for expression of the novel phenotype. By virtue ofthe high transformation efficiency, successful transformations can beachieved within reasonable time periods and without unduly repetitiveprocedures.

All patents, other publications, and patent applications mentioned aboveare illustrative of the skill of those skilled in the art to which theinvention pertains. Each patent, other publication and patentapplication is herein individually incorporated by reference in the samelocation and to the same extent as if each patent, other publication, orpatent application had been individually indicated to be incorporated byreference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. Transgenic Brassica species cells and progenythereof comprising an expression cassette, wherein said cells arecharacterized as oncogene-free and capable of regeneration tomorphologically normal whole plants, and wherein said expressioncassette comprises, in the 5'-3' direction of transcription:(1) atranscription initiation region functional in Brassica species cells;(2) a DNA sequence comprising an open reading frame having an initiationcodon at its 5' terminus or a nucleic acid sequence complementary to anendogenous transcription product which when expressed will alter thephenotype of said transgenic cells; (3) a transcription terminationregion functional in Brassica species cells; (4) a right border ofT-DNA; and (5) a structural gene capable of expression in said Brassicaproviding for selection of transgenic Brassica species cells; whereinsaid expression cassette is capable of altering the phenotype of saidBrassica species cells when said cells are grown under conditionswhereby said DNA sequence or said nucleic acid sequence is expressed. 2.Cells according to claim 1, wherein said Brassica is napus orcampestris.
 3. Cells according to claim 1, wherein said transcriptioninitiation region is the 35S region of cauliflower mosaic virus.
 4. ABrassica plant comprising cells according to claim
 1. 5. A Brassicaplant according to claim 4, wherein said Brassica is of the speciescampestris or napus.
 6. A cell culture of cells according to claim
 1. 7.A cell culture of cells according to claim 6, wherein said culture iscapable of growth in a culture medium which includes a selective agentto which said structural gene provides resistance.
 8. A transformedBrassica plant produced according to the methodcomprising:co-cultivating Brassica cells with disarmed A. tumefacienscomprising a plasmid containing an insertion sequence resulting fromjoining in vitro of a transcription cassette to at least the right T-DNAborder of a Ti- or Ri-plasmid whereby said Brassica cells aretransformed with said insertion sequence which becomes integrated intothe plant cell genome to provide transformed oncogene-free cellstransferring said transformed oncogene-free cells to callus inducingmedium, wherein said callus inducing medium contains at least one auxinand a means for selecting for transformed cells as a result of a markercarried on said plasmid whereby callus comprising transformed cells isproduced; transferring said callus to regeneration medium containingless than about 2% sucrose or an organic caloric equivalent thereto toproduce shoots; and transferring said shoots to a growing medium toproduce plants capable of having an altered phenotype when grown undercondition whereby a DNA sequence in said insertion sequence isexpressed.
 9. A plant according to claim 8, wherein said transcriptioncassette comprises the cauliflower mosaic virus 35S promoter.